Noritada Kaji

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid

Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEG-unmodified MEND due to less accumulation in liver and spleen.

Nanospheres for DNA separation chips

We report here a technology to carry out separations of a wide range of DNA fragments with high speed and high resolution. The approach uses a nanoparticle medium, core-shell type nanospheres, in conjunction with a pressurization technique during microchip electrophoresis. DNA fragments up to 15 kilobase pairs (kbp) were successfully analyzed within 100 s without observing any saturation in migration rates. DNA fragments migrate in the medium while maintaining their characteristic molecular structure. To guarantee effective DNA loading and electrofocusing in the nanosphere solution, we developed a double pressurization technique. Optimal pressure conditions and concentrations of packed nanospheres are critical to achieve improved DNA separations.

Trafficking and Subcellular Localization of Multiwalled Carbon Nanotubes in Plant Cells

Major barriers to delivery of biomolecules are crossing the cellular membranes and achieving a high cytoplasmic concentration by circumventing entrapment into endosomes and other lytic organelles. Motivated by such aim, we have investigated the capability of multiwalled carbon nanotubes (MWCNTs) to penetrate the cell membrane of plant protoplasts (plant cells made devoid of their cell walls via enzymatic treatment) and studied their internalization mechanism via confocal imaging and TEM techniques. Our results indentified an endosome-escaping uptake mode of MWCNTs by plant protoplasts. Moreover, short MWCNTs (<100 nm) were observed to target specific cellular substructures including the nucleus, plastids, and vacuoles. These findings are expected to have a significant impact on plant cell biology and transformation technologies.

Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells.

Quantum dots (QDs) have been used to study the effects of fluorescent probes for biomolecules and cell imaging. Adipose tissue-derived stem cells, which carry a relatively lower donor site morbidity, while yielding a large number of stem cells at harvest, were transduced with QDs using the octa-arginine peptide (R8) cell-penetrating peptide (CPP). The concentration ratio of QDs:R8 of 1 x 10(4) was optimal for delivery into ASCs. No cytotoxicity was observed in ASCs transduced with less than 16 nM of QDs655. In addition, >80% of the cells could be labeled within 1 h and the fluorescent intensity was maintained at least for 2 weeks. The ASCs transduced with QDs using R8 could be differentiated into both adipogenic and osteogenic cells, thus suggesting that the cells maintained their stem cell potency. The ASCs labeled with QDs using R8 were further transplanted subcutaneously into the backs of mice or into mice through the tail vein. The labeled ASCs could be imaged with good contrast using the Maestro in vivo imaging system. These data suggested that QD labeling using R8 could be utilized for the imaging of ASCs.

Size-Selective Growth and Stabilization of Small CdSe Nanoparticles in Aqueous Solution

Using cysteine and its derivatives as capping molecules, we investigated the influence of the physical structure and chemical nature of capping molecules on the selective growth and stabilization of small CdSe nanoparticles (NPs) in aqueous solution at room temperature. Our investigations revealed specific roles for each functional group of cysteine, and we could correlate this structure and nature of the capping molecules with the size, size restriction, size distribution, and stability of the NPs. For selective growth and stabilization of the NPs in aqueous solution, their capping molecules should have at least one functional group with strong nucleophilicity as well as another free, charged functional group. Capping molecules acting as a monodentate ligand were more effective than those acting as a bidentate ligand for restricting the NPs to a smaller size, whereas the former was less effective than the latter for getting a narrower NP size distribution. Capping molecules with relatively bulky spatial geometry near the ligand-NP interface resulted in the formation of NPs with poor short- and long-term stabilities, whereas those having relatively compact spatial geometry near the interface led to NPs with at least moderate short-term stability. We saw that capping molecules having relatively compact outermost spatial geometry led to NPs with excellent long-term stability, whereas those having relatively bulky outermost spatial geometry produced NPs with at most only moderate long-term stability. Our results clearly showed general trends for the possibility of selective growth of stable semiconductor NPs with particular sizes in aqueous solution.

Monitoring transplanted adipose tissue-derived stem cells combined with heparin in the liver by fluorescence imaging using quantum dots.

Adipose tissue-derived stem cell (ASC) transplantation, when used in combination with heparin, has proven to be an effective treatment for acute liver failure in mice. However, the behavior and organ-specific accumulation of transplanted ASCs alone or in combination with heparin is poorly understood. In this paper, we investigated whether quantum dots (QDs) labeling using octa-arginine peptide (R8) for ASCs could be applied for in vivo fluorescence imaging in mice with acute liver failure, and analyzed the behavior and organ-specific accumulation of ASCs that were transplanted alone or in combination with heparin using an IVIS(®) Spectrum analysis. Almost all of the transplanted ASCs were observed to accumulate in the lungs within 10 min without heparin. However, when heparin was used in combination with the ASCs, the accumulation of the transplanted ASCs was found not only in the lungs but also in the liver. The region of interest (ROI) analysis of ex vivo fluorescence imaging showed that the accumulation rate of transplanted ASCs in the liver increased to about 30%. In the time course analysis, the accumulation rate of ASCs in the liver was about 10% in 1 day and was maintained at that level for at least 2 day. We observed that heparin was effective for increasing the accumulation of transplanted ASCs in the liver using fluorescence imaging technology. We suggest that fluorescence imaging by means of QDs labeling using R8 can be useful for tracing the transplanted cells.

Simultaneous separation, metering, and dilution of plasma from human whole blood in a microfluidic system.

In about a 3 min period, we have simultaneously separated plasma from human whole blood and metered and diluted the plasma using a microchip with an interchannel microstructure. The plasma separation was based on both cross-flow filtration and sedimentation of red blood cells in the microchannels. Metering and diluting operations of the plasma were based on volume control of liquid in the microchannels by syringe pumps. On this microchip, we produced plasma diluted by a factor of 6 from whole blood containing theophylline and we observed very little hemolysis. It is possible to separate plasma from one or just several drops of whole blood by using this microchip.

Rolling circle amplification and circle-to-circle amplification of a specific gene integrated with electrophoretic analysis on a single chip.

We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37 degrees C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 min (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies.

Quantum dots for labeling adipose tissue-derived stem cells.

Adipose tissue-derived stem cells (ASCs) have a self-renewing ability and can be induced to differentiate into various types of mesenchymal tissue. Because of their potential for clinical application, it has become desirable to label the cells for tracing transplanted cells and for in vivo imaging. Quantum dots (QDs) are novel inorganic probes that consist of CdSe/ZnS-core/shell semiconductor nanocrystals and have recently been explored as fluorescent probes for stem cell labeling. In this study, negatively charged QDs655 were applied for ASCs labeling, with the cationic liposome, Lipofectamine. The cytotoxicity of QDs655-Lipofectamine was assessed for ASCs. Although some cytotoxicity was observed in ASCs transfected with more than 2.0 nM of QDs655, none was observed with less than 0.8 nM. To evaluate the time dependency, the fluorescent intensity with QDs655 was observed until 24 h after transfection. The fluorescent intensity gradually increased until 2 h at the concentrations of 0.2 and 0.4 nM, while the intensity increased until 4 h at 0.8 nM. The ASCs were differentiated into both adipogenic and osteogenic cells with red fluorescence after transfection with QDs655, thus suggesting that the cells retain their potential for differentiation even after transfected with QDs655. These data suggest that QDs could be utilized for the labeling of ASCs.